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Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency1. The α- and β-carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)—a complex of eight large (RbcL) and eight small (RbcS) subunits—and carbonic anhydrase2–4. As HCO3− can diffuse through the proteinaceous carboxysome shell but CO2 cannot5, carbonic anhydrase generates high concentrations of CO2 for carbon fixation by Rubisco6. The shell also prevents access to reducing agents, generating an oxidizing environment7–9. The formation of β-carboxysomes involves the aggregation of Rubisco by the protein CcmM10, which exists in two forms: full-length CcmM (M58 in Synechococcus elongatus PCC7942), which contains a carbonic anhydrase-like domain8 followed by three Rubisco small subunit-like (SSUL) modules connected by flexible linkers; and M35, which lacks the carbonic anhydrase-like domain11. It has long been speculated that the SSUL modules interact with Rubisco by replacing RbcS2–4. Here we have reconstituted the Rubisco–CcmM complex and solved its structure. Contrary to expectation, the SSUL modules do not replace RbcS, but bind close to the equatorial region of Rubisco between RbcL dimers, linking Rubisco molecules and inducing phase separation into a liquid-like matrix. Disulfide bond formation in SSUL increases the network flexibility and is required for carboxysome function in vivo. Notably, the formation of the liquid-like condensate of Rubisco is mediated by dynamic interactions with the SSUL domains, rather than by low-complexity sequences, which typically mediate liquid–liquid phase separation in eukaryotes12,13. Indeed, within the pyrenoids of eukaryotic algae, the functional homologues of carboxysomes, Rubisco adopts a liquid-like state by interacting with the intrinsically disordered protein EPYC114. Understanding carboxysome biogenesis will be important for efforts to engineer CO2-concentrating mechanisms in plants15–19. The structure of a Rubisco–CcmM complex sheds light on the formation of carboxysomes in cyanobacteria.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO2 in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Rubisco is a remarkably inefficient enzyme, fixing only 2–10 CO2 molecules per second. Efforts to increase crop yields by bioengineering Rubisco remain unsuccessful, owing in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. The large subunit of Rubisco requires the chaperonin system for folding, and recent studies have shown that assembly of hexadecameric Rubisco is mediated by specific assembly chaperones. Moreover, Rubisco function can be inhibited by a range of sugar-phosphate ligands, including RuBP. Metabolic repair depends on remodeling of Rubisco by the ATP-dependent Rubisco activase and hydrolysis of inhibitory sugar phosphates by specific phosphatases. Here, we review our present understanding of the structure and function of these auxiliary factors and their utilization in efforts to engineer more catalytically efficient Rubisco enzymes.

Improving the performance of the key photosynthetic enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) by protein engineering is a critical strategy for increasing crop yields. The extensive chaperone requirement of plant Rubisco for folding and assembly has long been an impediment to this goal. Production of plant Rubisco in Escherichia coli requires the coexpression of the chloroplast chaperonin and four assembly factors. Here, we demonstrate that simultaneous expression of Rubisco and chaperones from a T7 promotor produces high levels of functional enzyme. Expressing the small subunit of Rubisco with a C-terminal hexahistidine-tag further improved assembly, resulting in a similar to 12-fold higher yield than the previously published procedure. The expression system described here provides a platform for the efficient production and engineering of plant Rubisco.

Proteins are composed of linear chains of amino acids. Upon synthesis in the cell, most proteins must rapidly acquire a specific three-dimensional structure, a process known as folding, before they can perform their various biological functions. Productive folding is often competed by aggregation, owing to the high macromolecular crowding of the cellular environment. Moreover, the process of translation increases the danger of misfolding, as incomplete nascent polypeptides are not yet able to fold into stable structures in many cases. To avoid these off-pathway reactions, a class of proteins called molecular chaperones has evolved in all organisms. They interact with nascent or stress-denatured polypeptides, prevent their aggregation and assist in folding and assembly processes, often in an ATP-regulated manner. These functions are especially important in conditions of cell stress, and their failure is linked with the manifestation of numerous age-dependent degenerative diseases. Key Concepts Molecular chaperones are proteins that mediate folding and assembly of other proteins, without being components of the final folded or assembled structures. Molecular chaperones prevent the potentially toxic aggregation of newly synthesised polypeptides during translation and when mature proteins unfold during stress conditions or ageing. Hsp70 chaperones act as a hub in the proteostasis network by assisting protein folding and conformational maintenance and by distributing client proteins to other chaperones for correct folding. The chaperonins mediate the folding of proteins with complex topologies and kinetically frustrated folding pathways. Keywords: protein folding; molecular chaperones; chaperonins; Hsp70; Hsp90; protein aggregation

A biotech tour de force RuBisCo, the key enzyme of photosynthesis, is a complex of eight large and eight small subunits. It mediates the fixation of atmospheric CO 2 in the Calvin-Benson-Bassham cycle. In addition to being enzymatically inefficient, RuBisCo has a problem with distinguishing between CO 2 and O 2 . The fixation of O 2 results in the energetically wasteful reaction of photorespiration. Thus, there is a strong incentive to improve RuBisCo's catalytic properties by engineering. However, for decades, it has been impossible to express the enzyme from plants in an easily manipulatable bacterial host. Aigner et al. succeeded in functionally expressing plant RuBisCo in Escherichia coli (see the Perspective by Yeates and Wheatley). This should allow for the systematic mutational analysis of RuBisCo and selection of favorable variants for improved crop yields. Science , this issue p. 1272 ; see also p. 1253

Rubisco, the enzyme that catalyzes the fixation of atmospheric CO(2) in photosynthesis, is subject to inactivation by inhibitory sugar phosphates. Here we report the 2.95-Å crystal structure of Nicotiana tabacum Rubisco activase (Rca), the enzyme that facilitates the removal of these inhibitors. Rca from tobacco has a classical AAA(+)-protein domain architecture. Although Rca populates a range of oligomeric states when in solution, it forms a helical arrangement with six subunits per turn when in the crystal. However, negative-stain electron microscopy of the active mutant R294V suggests that Rca functions as a hexamer. The residues determining species specificity for Rubisco are located in a helical insertion of the C-terminal domain and probably function in conjunction with the N-domain in Rubisco recognition. Loop segments exposed toward the central pore of the hexamer are required for the ATP-dependent remodeling of Rubisco, resulting in the release of inhibitory sugar.

The HspBP1 homolog Fes1p was recently identified as a nucleotide exchange factor (NEF) of Ssa1p, a canonical Hsp70 molecular chaperone in the cytosol of Saccharomyces cerevisiae. Besides the Ssa-type Hsp70s, the yeast cytosol contains three additional classes of Hsp70, termed Ssb, Sse and Ssz. Here, we show that Fes1p also functions as NEF for the ribosome-bound Ssb Hsp70s. Sequence analysis indicated that residues important for interaction with Fes1p are highly conserved in Ssa1p and Ssb1p, but not in Sse1p and Ssz1p. Indeed, Fes1p interacts with Ssa1p and Ssb1p with similar affinity, but does not form a complex with Sse1p. Functional analysis showed that Fes1p accelerates the release of the nucleotide analog MABA-ADP from Ssb1p by a factor of 35. In contrast to the interaction between mammalian HspBP1 and Hsp70, however, addition of ATP only moderately decreases the affinity of Fes1p for Ssb1p. Point mutations in Fes1p abolishing complex formation with Ssa1p also prevent the interaction with Ssb1p. The ATPase activity of Ssb1p is stimulated by the ribosome-associated complex of Zuotin and Ssz1p (RAC). Interestingly, Fes1p inhibits the stimulation of Ssb1p ATPase by RAC, suggesting a complex regulatory role of Fes1p in modulating the function of Ssb Hsp70s in co-translational protein folding.

HspBP1 belongs to a family of eukaryotic proteins recently identified as nucleotide exchange factors for Hsp70. We show that the S. cerevisiae ortholog of HspBP1, Fes1p, is required for efficient protein folding in the cytosol at 37°C. The crystal structure of HspBP1, alone and complexed with part of the Hsp70 ATPase domain, reveals a mechanism for its function distinct from that of BAG-1 or GrpE, previously characterized nucleotide exchange factors of Hsp70. HspBP1 has a curved, all α-helical fold containing four armadillo-like repeats unlike the other nucleotide exchange factors. The concave face of HspBP1 embraces lobe II of the ATPase domain, and a steric conflict displaces lobe I, reducing the affinity for nucleotide. In contrast, BAG-1 and GrpE trigger a conserved conformational change in lobe II of the ATPase domain. Thus, nucleotide exchange on eukaryotic Hsp70 occurs through two distinct mechanisms.

The chaperonin GroEL has an essential role in mediating protein folding in the cytosol of Escherichia coli. Here we show that GroEL interacts strongly with a well-defined set of approximately 300 newly translated polypeptides, including essential components of the transcription/translation machinery and metabolic enzymes. About one third of these proteins are structurally unstable and repeatedly return to GroEL for conformational maintenance. GroEL substrates consist preferentially of two or more domains with alphabeta-folds, which contain alpha-helices and buried beta-sheets with extensive hydrophobic surfaces. These proteins are expected to fold slowly and be prone to aggregation. The hydrophobic binding regions of GroEL may be well adapted to interact with the non-native states of alphabeta-domain proteins.

A role for DnaK, the major E. coli Hsp70, in chaperoning de novo protein folding has remained elusive. Here we show that under nonstress conditions DnaK transiently associates with a wide variety of nascent and newly synthesized polypeptides, with a preference for chains larger than 30 kDa. Deletion of the nonessential gene encoding trigger factor, a ribosome-associated chaperone, results in a doubling of the fraction of nascent polypeptides interacting with DnaK. Combined deletion of the trigger factor and DnaK genes is lethal under normal growth conditions. These findings indicate important, partially overlapping functions of DnaK and trigger factor in de novo protein folding and explain why the loss of either chaperone can be tolerated by E. coli.

The cylindrical chaperonin GroEL and its cofactor GroES mediate ATP-dependent protein folding in Escherichia coli . Recent studies in vitro demonstrated that GroES binding to GroEL causes the displacement of unfolded polypeptide into the central volume of the GroEL cavity for folding in a sequestrated environment. Resulting native protein leaves GroEL upon GroES release, whereas incompletely folded polypeptide can be recaptured for structural rearrangement followed by another folding trial. Additionally, each cycle of GroES binding and dissociation is associated with the release of nonnative polypeptide into the bulk solution. Here we show that this loss of substrate from GroEL is prevented when the folding reaction is carried out in the presence of macromolecular crowding agents, such as Ficoll and dextran, or in a dense cytosolic solution. Thus, the release of nonnative polypeptide is not an essential feature of the productive chaperonin mechanism. Our results argue that conditions of excluded volume, thought to prevail in the bacterial cytosol, increase the capacity of the chaperonin to retain nonnative polypeptide throughout successive reaction cycles. We propose that the leakiness of the chaperonin system under physiological conditions is adjusted such that E. coli proteins are likely to complete folding without partitioning between different GroEL complexes. Polypeptides that are unable to fold on GroEL eventually will be transferred to other chaperones or the degradation machinery.

The role of the abundant stress protein Hsp90 in protecting cells against stress-induced damage is not well understood. The recent discovery that a class of ansamycin antibiotics bind specifically to Hsp90 allowed us to address this problem from a new angle. We find that mammalian Hsp90, in cooperation with Hsp70, p60, and other factors, mediates the ATP-dependent refolding of heat-denatured proteins, such as firefly luciferase. Failure to refold results in proteolysis. The ansamycins inhibit refolding, both in vivo and in a cell extract, by preventing normal dissociation of Hsp90 from luciferase, causing its enhanced degradation. This mechanism also explains the ansamycin-induced proteolysis of several protooncogenic protein kinases, such as Raf-1, which interact with Hsp90. We propose that Hsp90 is part of a quality control system that facilitates protein refolding or degradation during recovery from stress. This function is used by a limited set of signal transduction molecules for their folding and regulation under nonstress conditions. The ansamycins shift the mode of Hsp90 from refolding to degradation, and this effect is probably amplified for specific Hsp90 substrates.

As a basic principle, assisted protein folding by GroEL has been proposed to involve the disruption of misfolded protein structures through ATP hydrolysis and interaction with the cofactor GroES. Here, we describe chaperonin subreactions that prompt a re-examination of this view. We find that GroEL-bound substrate polypeptide can induce GroES cycling on and off GroEL in the presence of ADP. This mechanism promotes efficient folding of the model protein rhodanese, although at a slower rate than in the presence of ATP. Folding occurs when GroES displaces the bound protein into the sequestered volume of the GroEL cavity. Resulting native protein leaves GroEL upon GroES release. A single-ring variant of GroEL is also fully functional in supporting this reaction cycle. We conclude that neither the energy of ATP hydrolysis nor the allosteric coupling of the two GroEL rings is directly required for GroEL/GroES-mediated protein folding. The minimal mechanism of the reaction is the binding and release of GroES to a polypeptide-containing ring of GroEL, thereby closing and opening the GroEL folding cage. The role of ATP hydrolysis is mainly to induce conformational changes in GroEL that result in GroES cycling at a physiologically relevant rate.

Folding of newly synthesized polypeptides in the crowded cellular environment requires the assistance of so-called molecular chaperone proteins. Chaperones of the Hsp70 class and their partner proteins interact with nascent polypeptide chains on ribosomes and prevent their premature (mis)folding at least until a domain capable of forming a stable structure is synthesized. For many proteins, completion of folding requires the subsequent interaction with one of the large oligomeric ring-shaped proteins of the chaperonin family, which is composed of the GroEL-like proteins in eubacteria, mitochondria, and chloroplasts, and the TRiC family in eukaryotic cytosol and archaea. These proteins bind partially folded polypeptide in their central cavity and promote folding by ATP-dependent cycles of release and rebinding. In these reactions, molecular chaperones interact predominantly with the hydrophobic surfaces exposed by nonnative polypeptides, thereby preventing incorrect folding and aggregation.

The chaperonins GroEL and GroES of Escherichia coli facilitate protein folding in an adenosine triphosphate (ATP)-dependent reaction cycle. The kinetic parameters for the formation and dissociation of GroEL-GroES complexes were analyzed by surface plasmon resonance. Association of GroES and subsequent ATP hydrolysis in the interacting GroEL toroid resulted in the formation of a stable GroEL:ADP:GroES complex. The complex dissociated as a result of ATP hydrolysis in the opposite GroEL toroid, without formation of a symmetrical GroEL:(GroES)2 intermediate. Dissociation was accelerated by the addition of unfolded polypeptide. Thus, the functional chaperonin unit is an asymmetrical GroEL:GroES complex, and substrate protein plays an active role in modulating the chaperonin reaction cycle.

The Escherichia coli chaperonin GroEL and its regulator GroES are thought to mediate adenosine triphosphate-dependent protein folding as an asymmetrical complex, with substrate protein bound within the GroEL cylinder. In contrast, a symmetrical complex formed between one GroEL and two GroES oligomers, with substrate protein binding to the outer surface of GroEL, was recently proposed to be the functional chaperonin unit. Electron microscopic and biochemical analyses have now shown that unphysiologically high magnesium concentrations and increased pH are required to assemble symmetrical complexes, the formation of which precludes the association of unfolded polypeptide. Thus, the functional significance of GroEL:(GroES)2 particles remains to be demonstrated.

The conformation of a three-disulphide derivative of bovine alpha-lactalbumin bound to the molecular chaperone GroEL has been investigated by monitoring directly its hydrogen exchange kinetics using electrospray ionization mass spectrometry. The bound protein is weakly protected from exchange to an extent closely similar to that of an uncomplexed molten globule state of the three-disulphide protein. Binding to GroEL in this system appears to involve relatively disordered partly folded states resembling intermediates formed in the very early stages of kinetic folding of many proteins in vitro.

Protein folding in mitochondria is mediated by the chaperonin Hsp60, the homologue of E. coli GroEL. Mitochondria also contain a homologue of the cochaperonin GroES, called Hsp10, which is a functional regulator of the chaperonin. To define the in vivo role of the co-chaperonin, we have used the genetic and biochemical potential of the yeast S. cerevisiae. The HSP10 gene was cloned and sequenced and temperature-sensitive lethal hsp10 mutants were generated. Our results identify Hsp10 as an essential component of the mitochondrial protein folding apparatus, participating in various aspects of Hsp60 function. Hsp10 is required for the folding and assembly of proteins imported into the matrix compartment, and is involved in the sorting of certain proteins, such as the Rieske Fe/S protein, passing through the matrix en route to the intermembrane space. The folding of the precursor of cytosolic dihydrofolate reductase (DHFR), imported into mitochondria as a fusion protein, is apparently independent of Hsp10 function consistent with observations made for the chaperonin-mediated folding of DHFR in vitro. The temperature-sensitive mutations in Hsp10 map to a domain (residues 25-40) that corresponds to a previously identified mobile loop region of bacterial GroES and result in a reduced binding affinity of hsp10 for the chaperonin at the non-permissive temperature.

Molecular chaperones are a class of proteins that interact with the non-native conformations of other proteins. The major role of chaperones of the Hsp70 and Hsp60 families is to prevent aggregation of newly synthesized polypeptides and then to mediate their folding to the native state. As a result of functional studies of these proteins, there has been a revision of the long-held view that protein folding in the cell is a spontaneous process.

THE chaperonin GroEL, a tetradecameric cylinder consisting of subunits of Mr∼60,000 (60K), and its cofactor GroES, a heptameric ring of 10K subunits, mediate protein folding in the cytosol of Escherichia coll1–3. In the presence of nucleotide, GroES forms a 1:1 complex with GroEL which binds unfolded protein in its central cavity and releases it to allow folding upon ATP hydrolysis4–7. Using labelling with azido-ATP, we have identified a protease-stable nucleotide-binding domain of Mr 40K in the GroEL subunits (residues 153-531). Azido-ATP is crosslinked to the highly conserved Tyr 477, indicating that this residue is close to the purine ring of the bound nucleotide. Surprisingly, GroES also binds ATP cooperatively and with an affinity comparable to that of GroEL. Azido-nucleotide labelling of GroES subunits occurs at the conserved Tyr 71 in a protease-stable 6.5K domain (starting at residue 33). Proteinase K cleavage at residue 32 is prevented when GroES is bound to GroEL. ATP binding to GroES may be important in charging the seven subunits of the interacting GroEL ring with ATP to facilitate cooperative ATP binding and hydrolysis for substrate protein release.

3. HSP70 PROTEINS: CHAPERONES WITH DIVERSE ROLES IN PROTEIN METABOLISM . . .. . . . . . . . . .. . . . . .. . . ... . . . .... . . . . 357 Structure and Function of Hsp70 Chaperones . . . . . . . . . . . . . . . . . . . . . . . 357 Maintenance of the Translocation-Competent State of Precursor Proteins . . . . . . 359 The Hsp70 DnaK and Dna! are a Chaperone Team . . . . . . . . . . . . . . . . . . . 360 Organellar Hsp70s in Membrane Translocation and Folding .. . . . .. . . . . . . 361 Hsp70 in Cells Under Metabolic Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 Hsp70 and Protein Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

We used peroxisomal alcohol oxidase (AO) for the affinity purification of molecular chaperones from yeasts. Methodical studies showed that up to 0.8 mg of purified bacterial GroEL was able to bind per ml of immobilized denatured AO column material. Using crude extracts of Hansenula polymorpha or Saccharomyces cerevisiae, several proteins were specifically eluted with Mg-ATP which were recognized by antibodies against hsp60 or hsp70. One H. polymorpha 70 kDa protein was strongly induced during growth at elevated temperatures, whereas a second 70 kDa protein as well as a 60 kDa protein showed strong protein sequence homology to mitochondrial SSCI and hsp60, respectively, from S. cerevisiae.

The mechanism of GroEL (chaperonin)-mediated protein folding is only partially understood. We have analysed structural and functional properties of the interaction between GroEL and the co-chaperonin GroES. The stoichiometry of the GroEL 14mer and the GroES 7mer in the functional holo-chaperonin is 1:1. GroES protects half of the GroEL subunits from proteolytic truncation of the approximately 50 C-terminal residues. Removal of this region results in an inhibition of the GroEL ATPase, mimicking the effect of GroES on full-length GroEL. Image analysis of electron micrographs revealed that GroES binding triggers conspicuous conformational changes both in the GroES adjacent end and at the opposite end of the GroEL cylinder. This apparently prohibits the association of a second GroES oligomer. Addition of denatured polypeptide leads to the appearance of irregularly shaped, stain-excluding masses within the GroEL double-ring, which are larger with bound alcohol oxidase (75 kDa) than with rhodanese (35 kDa). We conclude that the functional complex of GroEL and GroES is characterized by asymmetrical binding of GroES to one end of the GroEL cylinder and suggest that binding of the substrate protein occurs within the central cavity of GroEL.

T-complex polypeptide 1 (TCP-1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP-1 to be part of a hetero-oligomeric 970 kDa complex containing several structurally related subunits of 52-65 kDa. These members of a new protein family are assembled into a TCP-1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP-dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co-chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.

The increased synthesis of heat shock proteins is a ubiquitous physiological response of cells to environmental stress. How these proteins function in protecting cellular structures is not yet understood. The mitochondrial heat shock protein 60 (Hsp60) has now been shown to form complexes with a variety of polypeptides in organelles exposed to heat stress. The Hsp60 was required to prevent the thermal inactivation in vivo of native dihydrofolate reductase (DHFR) imported into mitochondria. In vitro, Hsp60 bound to DHFR in the course of thermal denaturation, preventing its aggregation, and mediated its adenosine triphosphate-dependent refolding at increased temperatures. These results suggest a general mechanism by which heat shock proteins of the Hsp60 family stabilize preexisting proteins under stress conditions.

There is evidence to suggest that components of archaebacteria are evolutionarily related to cognates in the eukaryotic cytosol. We postulated that the major heat-shock protein of the thermophilic archaebacterium, Sulfolobus shibatae, is a molecular chaperone and that it is related to an as-yet unidentified chaperone component in the eukaryotic cytosol. Acquired thermotolerance in S. shibatae correlates with the predominant synthesis of this already abundant protein, referred to as thermophilic factor 55 (TF55). TF55 is a homo-oligomeric complex of two stacked 9-membered rings, closely resembling the 7-membered-ring complexes of the chaperonins, groEL, hsp60 and Rubisco-binding protein. The TF55 complex binds unfolded polypeptides in vitro and has ATPase activity-features consistent with its being a molecular chaperone. The primary structure of TF55, however, is not significantly related to the chaperonins. On the other hand, it is highly homologous (36-40% identity) to a ubiquitous eukaryotic protein, t-complex polypeptide-1 (TCP1). In Saccharomyces cerevisiae, TCP1 is an essential protein that may play a part in mitotic spindle formation. We suggest that TF55 in archaebacteria and TCP1 in the eukaryotic cytosol are members of a new class of molecular chaperones.

Converging physiological, genetic, and biochemical studies have established the salient features of preprotein translocation across the plasma membrane of Escherichia coli. Translocation is catalyzed by two proteins, a soluble chaperone and a membrane-bound translocase. SecB, the major chaperone for export, forms a complex with preproteins. Complex formation inhibits side-reactions such as aggregation and misfolding and aids preprotein binding to the membrane surface. Translocase consists of functionally linked peripheral and integral membrane protein domains. SecA protein, the peripheral membrane domain of translocase, is the primary receptor for the SecB/preprotein complex. SecA hydrolyzes ATP, promoting cycles of translocation, preprotein release, Δµ~H+-dependent translocation, and rebinding of the preprotein. The membrane-embedded domain of translocase is the SecY/E protein. It has, as subunits, the SeeY and SecE polypeptides. The SecY/E protein stabilizes and activates SecA and participates in binding it to the membrane. SecA recognizes the leader domain of preproteins, whereas both SecA and SecB recognize the mature domain. Many proteins translocate without requiring SecB, and some proteins do not need translocase to assemble into the plasma membrane. Translocation is usually followed by endoproteolytic cleavage by leader peptidase. The availability of virtually every pure protein and cloned gene involved in the translocation process makes E. coli the premier organism for the study of translocation mechanisms.

Preprotein translocation in E. coli requires ATP, the membrane electrochemical potential Δμ H +, and translocase, an enzyme with an ATPase domain (SecA) and the membrane-embedded SecYE. Studies of translocase and proOmpA reveal a five-step catalytic cycle: First, proOmpA binds to the SecA domain. Second, SecA binds ATP. Third, ATP-binding energy permits translocation of ∼20 residues of proOmpA. Fourth, ATP hydrolysis releases proOmpA. ProOmpA may then rebind to SecA and reenter this cycle, allowing progress through a series of transmembrane intermediates. In the absence of Δμ H + or association with SecA, proOmpA passes backward through the membrane, but moves forward when either ATP and SecA or a membrane electrochemical potential is supplied. However, in the presence of Δμ H + (fifth step), proOmpA rapidly completes translocation. Δμ H + — driven translocation is blocked by SecA plus nonhydrolyzable ATP analogs, indicating that Δμ H + drives translocation when ATP and proOmpA are not bound to SecA.

The formation of insoluble protein aggregates in neurons is a hallmark of neurodegenerative diseases caused by proteins with expanded polyglutamine (polyQ) repeats. However, the mechanistic relationship between polyQ aggregation and its toxic effects on neurons remains unclear. Two main hypotheses have been put forward for how polyQ expansions may cause cellular dysfunction. In one model neurotoxicity results from the ability of polyQ-expanded proteins to recruit other important cellular proteins with polyQ stretches into the aggregates. In the other model, aggregating polyQ proteins partially inhibit the ubiquitin–proteasome system for protein degradation. These two mechanisms are not exclusive but may act in combination. In general, protein misfolding and aggregation are prevented by the machinery of molecular chaperones. Some chaperones such as the members of the Hsp70 family also modulate polyQ aggregation and suppress its toxicity. These recent findings suggest that an imbalance between the neuronal chaperone capacity and the production of potentially dangerous polyQ proteins may trigger the onset of polyQ disease.

Heat shock proteins (Hsps) and molecular chaperones isolated from tumors or virally infected cells elicit an efficient CD8+ T cell response against bound antigenic peptides. This immune response is mediated by presentation of the peptides on MHC class I complexes of antigen-presenting cells (APCs), but the cellular mechanism of this presentation process is not yet understood. Here we provide evidence for the existence of a proteinaceous receptor on the surface of APCs that is specific for mammalian Hsp70. Using a flow cytometry-based assay, saturable binding of Hsp70 to the cell surface of macrophages and peripheral blood monocytes, but not of lymphocytes, can be demonstrated. The affinity of the receptor is in the sub-micromolar range (Kd < 100 nM). Only mammalian Hsc70/Hsp70, but not bacterial Hsp70, is bound with high affinity. Subsequent to binding, Hsp70 is taken up by endocytosis, resulting in an intracellular localization. Our results suggest that receptor-mediated endocytosis forms the basis for the demonstrated efficacy of Hsp70-peptide complexes as anti-tumor vaccines.

The cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel with 12 membrane-spanning sequences, undergoes inefficient maturation in the endoplasmic reticulum (ER). Potentially charged residues in transmembrane segments may contribute to this defect in biogenesis. We demonstrate that transmembrane segment 6 of CFTR, which contains three basic amino acids, is extremely unstable in the lipid bilayer upon membrane insertion in vitro and in vivo. However, two distinct mechanisms counteract this anchoring deficiency: (i) the ribosome and the ER translocon co-operate to prevent transmembrane segment 6 from passing through the membrane co- translationally; and (ii) cytosolic domains of the ion channel post-translationally maintain this segment of CFTR in a membrane-spanning topology. Although these mechanisms are essential for successful completion of CFTR biogenesis, inefficiencies in their function retard the maturation of the protein. It seems possible that some of the disease-causing mutations in CFTR may reduce the efficiency of proper membrane anchoring of the protein.

The cylindrical chaperonin GroEL of E. coli and its ring-shaped cofactor GroES cooperate in mediating the ATP-dependent folding of a wide range of polypeptides in vivo and in vitro. By binding to the ends of the GroEL cylinder, GroES displaces GroEL-bound polypeptide into an enclosed folding cage, thereby preventing protein aggregation during folding. The dynamic interaction of GroEL and GroES is regulated by the GroEL ATPase and involves the formation of asymmetrical GroEL:GroES1 and symmetrical GroEL: GroES2 complexes. The proposed role of the symmetrical complex as a catalytic intermediate of the chaperonin mechanism has been controversial. It has also been suggested that the formation of GroEL:GroES2 complexes allows the folding of two polypeptide molecules per GroEL reaction cycle, one in each ring of GroEL. By making use of a procedure to stabilize chaperonin complexes by rapid crosslinking for subsequent analysis by native PAGE, we have quantified the occurrence of GroEL:GroES1 and GroEL:GroES2 complexes in active refolding reactions under a variety of conditions using mitochondrial malate dehydrogenase (mMDH) as a substrate. Our results show that the symmetrical complexes are neither required for chaperonin function nor does their presence significantly increase the rate of mMDH refolding. In contrast, chaperonin-assisted folding is strictly dependent on the formation of asymmetrical GroEL:GroES1 complexes. These findings support the view that GroEL:GroES2 complexes have no essential role in the chaperonin mechanism.

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.

The effects of the human DnaJ homolog, Hsp40, on the ATPase and chaperone functions of the constitutively expressed Hsp70 homolog, Hsc70, were analyzed. Hsp40 stimulates the hydrolysis of ATP by Hsc70, causing a approximately 7-fold increase in its steady-state ATPase activity. In contrast to the prokaryotic Hsp70 system, ATP-hydrolysis and not the release of bound ADP is the rate-limiting step in the overall ATPase cycle of mammalian Hsc70. The ability to activate the Hsc70 ATPase is partially preserved in a deletion mutant containing the J-domain and the G/F region of Hsp40 but not in a deletion mutant that contains the J-domain alone. As a result of its ATPase stimulating activity, addition of Hsp40 allows Hsc70 to bind peptide in the presence of ATP, whereas in the absence of Hsp40, peptide is efficiently released upon ATP binding to Hsc70. The functional cooperation of Hsp40 with Hsc70 is essential to ensure the ATP hydrolysis-dependent binding of aggregation-sensitive denatured polypeptides, such as thermally denatured firefly luciferase and chemically denatured rhodanese. Binding of these proteins results in the formation of ternary complexes of Hsc70, Hsp40, and substrates. Hsc70 and Hsp40 cooperate with further factors in protein renaturation, as demonstrated by the finding that luciferase, thermally denatured in the presence of Hsc70, Hsp40, and ATP, refolds upon addition of rabbit reticulocyte cytosol. Our results indicate that Hsp40 has a critical regulatory function in the Hsc70 ATPase cycle that is required for the efficient loading of peptide substrate onto Hsc70.

The folding of many newly synthesized proteins in the cell depends on a set of conserved proteins known as molecular chaperones. These prevent the formation of misfolded protein structures, both under normal conditions and when cells are exposed to stresses such as high temperature. Significant progress has been made in the understanding of the ATP-dependent mechanisms used by the Hsp70 and chaperonin families of molecular chaperones, which can cooperate to assist in folding new polypeptide chains.

USA Understanding the mechanisms and pathways of protein folding constitutes a problem of fundamental biological significance. It has been known for more than three decades that all the information required for the acqisition of the native state is contained in the linear amino acid sequence of the polypeptide chain. Proteins are capable of spontaneous folding in the test-tube, at least under carefully chosen conditions, and this has led to the view that also within cells newly-synthesized polypeptides reach their native state in an essentially spontaneous reaction. Only more recently has it been realized that this is not generally the case. Cells contain a complex machinery of proteins, folding catalysts and so-called molecular chaperones, which mediate folding in the cytosol as well as within subcellular compartments such as mitochondria, chloroplasts and the endoplasmic reticulum (Hendrick and Hartl, 1993; Hard et al. , 1994; Hartl and Martin, 1995). Molecular chaperones, mostly constitutively expressed stress proteins, play a preeminent role in these processes and are the main focus of this chapter.

Recent evidence supports the view that cellular protein folding may be mediated by molecular chaperones. A fundamental question concerns the stage in its biogenesis at which the folding protein makes first contact with these components. We show here by crosslinking that the chaperone DnaJ binds nascent ribosome-bound polypeptide chains as short as 55 residues. Cotranslational binding of DnaJ to firefly luciferase and chloramphenicol acetyltransferase resulted in an arrest of folding as long as the functional partners of DnaJ in Escherichia coli, DnaK and GrpE, were missing. Protein uptake into microsomes and mitochondria was also interrupted by DnaJ. Both folding and post-translational translocation recommenced upon addition of DnaK and GrpE. We propose that DnaJ protects nascent polypeptide chains against aggregation and, in cooperation with Hsp70, controls their productive folding once a complete polypeptide or a polypeptide domain has been synthesized.

Proteins generally must fold into precise three-dimensional conformations to fulfill their biological functions. In the cell, this fundamental process is aided by molecular chaperones, which act in preventing protein misfolding and aggregation. How this machinery assists newly synthesized polypeptide chains in navigating the complex folding energy landscape is now being understood in considerable detail. The mechanisms that ensure the maintenance of a functional proteome under normal and stress conditions are also of great medical relevance, as the aggregation of proteins that escape the cellular quality control underlies a range of debilitating diseases, including many age-of-onset neurodegenerative disorders.

The main stress proteins of Escherichia coli function in an ordered protein-folding reaction. DnaK (heat-shock protein 70) recognizes the folding polypeptide as an extended chain and cooperates with DnaJ in stabilizing an intermediate conformational state lacking ordered tertiary structure. Dependent on GrpE and ATP hydrolysis, the protein is then transferred to GroEL (heat-shock protein 60) which acts catalytically in the production of the native state. This sequential mechanism of chaperone action may represent an important pathway for the folding of newly synthesized polypeptides.

THE MEMBERS OFTHE HSP70 FAMILY •• 298 Unfolding Is Required for Membrane Translocation of Proteins 299 Cytoso/ic Hsp70 Stabilizes Precursor Proteins for Translocation 300 Organellar Hsp70 in Protein Translocation and Folding 301 Requirement of Hsp70 for Protein Assembly in the ER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 Molecular Mechanism of Hsp70 Action .... .. . . . . . . . . . .. . .. . . .. ... . . . ........ 303 THE MEMBERS OF THE HSP60 FAMILY ... ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 306 Multiple Functions of Mitochondrial Hsp60 308 Molecular Mechanism of Chaperon in Action . .. . . . 311

Folding of two monomeric enzymes mediated by groE has been reconstituted in vitro. The groEL protein stabilizes the polypeptides in a conformation resembling the 'molten globule' state. Mg-ATP and groES then promote the acquisition of ordered tertiary structure at the surface of groEL. Folding requires the hydrolysis of about 100 ATP molecules per protein monomer. This active process of surface-mediated chain folding might represent a general mechanism for the formation of protein structure in vivo.

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.

ATP is involved in conferring transport competence to numerous mitochondrial precursor proteins in the cytosol. Unfolded precursor proteins were found not to require ATP for import into mitochondria, suggesting a role of ATP in the unfolding of precursors. Here we report the unexpected finding that a hybrid protein containing the tightly folded passenger protein dihydrofolate reductase becomes unfolded and specifically translocated across the mitochondrial membranes independently of added ATP. Moreover, interaction of the precursor with the mitochondrial receptor components does not require ATP. The results suggest that ATP is not involved in the actual process of unfolding during membrane translocation of precursors. ATP rather appears to be necessary for preventing the formation of improper structures of precursors in the cytosol and for folding of imported polypeptides on (and release from) chaperone-like molecules in the mitochondrial matrix.

Two distinct pathways of sorting and assembly of nuclear-encoded mitochondrial inner membrane proteins are described. In the first pathway, precursor proteins that carry amino-terminal targeting signals are initially translocated via contact sites between both mitochondrial membranes into the mitochondrial matrix. They become proteolytically processed, interact with the 60-kDa heat-shock protein hsp60 in the matrix and are retranslocated to the inner membrane. The sorting of subunit 9 of Neurospora crassa Fo-ATPase has been studied as an example. Fo subunit 9 belongs to that class of nuclear-encoded mitochondrial proteins which are evolutionarily derived from a prokaryotic ancestor according to the endosymbiont hypothesis. We suggest that after import into mitochondria, these proteins follow the ancestral sorting and assembly pathways established in prokaryotes (conservative sorting). On the other hand, ADP/ATP carrier was found not to require interaction with hsp60 for import and assembly. This agrees with previous findings that the ADP/ATP carrier possesses non-amino-terminal targeting signals and uses a different import receptor to other mitochondrial precursor proteins. It is proposed that the ADP/ATP carrier represents a class of mitochondrial inner membrane proteins which do not have a prokaryotic equivalent and thus appear to follow a non-conservative sorting pathway.

Mitochondrial heat-shock protein hsp60 functions in the folding of proteins imported into mitochondria. Folding occurs at the surface of hsp60 in an ATP-mediated reaction, followed by release of the bound polypeptides. We propose that hsp60 catalyses protein folding.